We previously described an alpha-amanitin-resistant mutant of D. melanogaster (AmaC4 or simply C4) with an altered, amanitin-resistant RNA polymerase II. We have now more fully characterized this mutant genetically and biochemically. We genetically mapped C4 to position 35.66 on the X chromosome and cytogenetically localized it to the polytene chromosome band interval 10C2-10D4. We then demonstrated that C4 is allelic to a previously known lethal-mutable locus I(1)L5 in this chromosomal region. Several known lethal alleles of L5 in fact affected the properties of RNA polymerase II in vitro. Following EMS mutagenesis of the C4-bearing chromosome we recovered new lethal L5 alleles, some of which were shown biochemically to have an altered amanitin-resistance polymerase II component. Furthermore, we induced mutants of C4 that had lost amanitin-resistance and showed that these mutants were also lethal alleles of L5. All the lethal alleles of L5 failed to completely complement each other genetically, and when analyzed biochemically their polymerase II displayed altered enzymatic properties. We conclude that C4 is an allele of the L5 locus and that this locus is most probably a structural gene for a subunit of RNA polymerase II. Some of the mutants at this locus display developmental abnormalities.